J.Bacchetta has documented that she has received grants for research activities from Amgen, Crinex, Sandoz.

J.Bacchetta has documented that she has received grants for research activities from Amgen, Crinex, Sandoz.

Epidemiological, immune and metabolic aspects of vitamin D in CKD Justine Bacchetta, MD, PhD

Calcium and phosphate metabolism 1-25 vitamin D Phosphorus Calcium FGF23 PTH Stimulating effect / Inhibiting effect Bacchetta, EMC 2015

Vitamin D Liver 25-hydroxylase 25OHD 1,25(OH) 2 D 1α-hydroxylase (CYP27B1) 24-hydroxylase (CYP24A1) Classical effects Hypercalcemia Hyperphosphatemia Decreased PTH levels Bone homeostasis VDR RXR 1,24,25(OH) 3 D RNA POL II 5 3 Target gene VDRE mrna expression Non classical and beneficial effects on global health Cancer Immunity Auto-immunity Inflammation Hypertension Prematurity

Epidemiological aspects of vitamin D in CKD

A U-curve between 25-D levels and mortality Sempos et al, JCEM 2013

Vitamin D and CKD A multi-scale impairment Cholecalciferol deficiency Inhibition of renal 1-α hydroxylase Downregulation by FGF23 Reduced endocytic putake by megalin/cubulin Increased activity of 24 hydroxylase Reduced sun exposure Reduces skin synthesis Reduced oral intake of vit D Proteinuria and loss of VDBP Loss in PD fluid Calcidiol deficiency Reduced availability of cholecalciferol Reduced liver synthesis of calcidiol Calcitriol deficiency Loss of VDR in parathyroid Impaired binding to VDR Calcitriol resistance Nigwekar, AJKD 2012

Vitamin D deficiency in pediatric CKD Author /country Year Patients Proportion of vitamine D deficiency (< 50 nmol/l) Proportion of vitamin D insufficiency (50-75 nmol/l) Menon, USA 2008 57 CKD II-IV 77 % Belostotsky, UK 2008 143 CKD 26 % 32 % Bacchetta, France 2010 227 CKD I-IV 40 % 40 % Cho, Korea 2013 59 CKD V-D 32 % 50 % Kumar, USA 2015 506 CKD from CKID 28 % Vitamin D levels depend on ethnicity Non-black Black Non-black Black Denburg Pediatric Nephrol 2013

Vitamin D levels depend on the underlying nephropathy in pediatric CKD 148 pediatric patients with CKD 2-5D Denburg Pediatric Nephrol 2013

Native vitamin D therapy and prevention of secondary hyperparathyroidism in pediatric CKD Randomized controlled trial Ergocalciferol versus placebo, 20 CKD 2-4 per group Shroff cjasn 2012

Immune aspects of vitamin D in CKD

Local synthesis of active vitamin D (monocytes) Pathogen TLR TLR DBP 25D 3 IL-15 + + IFNγ IL-4 1,25D 3 CYP27B1 + 1-25 vit D VDR LL37 = CAMP = cathelicin = antimicrobial peptide CYP24A1 LL37.... Bacterial killing Autophagosome Adapted from Adams & Hewison

fold-change in mrna fold-change mrna (relative to vehicle) fold-change in mrna fold-change mrna (relative to normal PD cells) Antibacterial responses by peritoneal macrophages are enhanced following vitamin D supplementation in pediatric patients undergoing PD In baseline PD cells In PD cells during an acute peritonitis (N=3 patients) 3 2 +25D *** 100 *** 10 * ** 1 0 CYP27B1 VDR CAMP HAMP * 1 0.1 * CYP27B1 VDR CYP24A1 CAMP HAMP 1000 100 +1,25D *** 100 CAMP HAMP ** ** 10 10 1 * 1 ** ** 0.1 CYP27B1 VDR CAMP HAMP 0.1 vehicle 25D 1,25D Bacchetta, PLOS One 2014

FGF23 is an inhibitor of extra-renal 1-alfa hydroxylase Fold change in mrna relative to vehicle-treated cells 1.20 1.00 0.80 0.60 0.40 0.20 0.00 * * * * CYP27B1 CYP24A1 VDR LL37 TNF FGF23: 100 ng/ml, 24 hours Controls = PBS 6 hours = black 24 hours = grey * * 25OHD 25OHD 25OHD Vehicle 1,25(OH) 2 D 1,25(OH) 2 D 1,25(OH) 2 D 1,25(OH) 2 D IL-15 1,25(OH) 2 D IL-15+ FGF23 1,25(OH) 2 D Bacchetta, JBMR 2012

High FGF23 levels and low 25-D levels: risk factors in the HEMO study Chonchol, JASN 2015

Metabolic aspects of vitamin D in CKD

Vitamin D, renin-angiotensin system and progressive renal impairment Chonchol Ann Med 2011 / Shroff Ped Nephrol 2012

Normal 25-D levels are associated with attenuated renal failure progression in children with CKD ESCAPE study 167 children (median egfr 51 ml/min per 1.73 m2) Shroff JASN 2015

25-D levels below 50 nmol/l as a risk factor for renal loss For 25D levels at baseline, during followup and when analysing with a mean 25D level ESCAPE study Shroff JASN 2015

25-D levels below 50 nmol/l are associated with greater diastolic blood pressure In healthy children In pediatric CKD 25 OH 20-50 50-75 >75 p (nmol/l) N 20 33 18 Systolic BP 111 104 106 0.021 (mmhg) Diastolic BP 66 60 59 0.032 (mmhg) PTH 21 19 16 0.021 VITADOS cohort 71 healthy children, 10-18 years Mean age 14±2 years No difference for age, anthropometric parameters, gender repartition and pubertal status Bacchetta, unpublished data ESCAPE study 167 children Median egfr 51 ml/min per 1.73 m2 Shroff JASN 2015

Icardi, NDT 2013 Vitamin D as a regulator of iron metabolism

serum 25D (ng/ml) serum 1,25D (pg/ml) serum hepcidin (ng/ml) Ergocalciferol decreases circulating hepcidin levels in vivo 25 OH vitamin D 1-25 OH 2 vitamin D Hepcidin 50 40 30 20 *** oral vitamin D 2 1 2 24 72 hrs baseline postvitamin D 60 50 40 30 20 oral vitamin D 2 1 2 24 72 hrs baseline postvitamin D 70 60 50 40 30 20 * ** oral vitamin D 2 1 2 24 72 hrs baseline postvitamin D *, p<0,05 in comparison to the first baseline 7 healthy volunteers 4 men, median age 42 yrs, range 27-63 yrs Bacchetta J, JASN 2014

Vitamin D decreases hepcidin and increases ferroportin in HepG2 cells and in PBMCs 1.2 fold-change mrna 1.0 0.8 0.6 0.4 0.2 * * 0.0 Vehicle 25OHD 1,25(OH) 2 D Ferritin/DAPI Ferritin ChIP analysis A decreased recruitment of RNA polymerase II after vitamin D on the hepcidin promoter Ethanol 25 OH-D 1-25 (OH)2-D Bacchetta J, JASN 2014

Vitamin D and bone metabolism: a strong inhibition of osteoclastogenesis % de cellules TRAP+ 3ny 100 ETH (contrôle) 25D (100nM) 1,25D (5nM) *** *** *** *** p<0.001 0 J0 J3-J6 J0-J3-J6 ETH 25D 1,25D Allard, Calcified Tissue International 2015

Pereira, Plos One 2015 Vitamin D and osteocytes in CKD 11 pediatric patients undergoing PD Bone biopsy before and after 8 months of doxercalciferol

Pereira, Plos One 2015 Vitamin D and osteocytes in CKD

Vitamin D and renal transplantation

Sub-optimal levels of vitamin D are frequent after renal Tx In adults 12% of patients are within the targets! N= 173 patients 7 years post Tx Similar data men/women But also in children N= 29 pediatric patients Versus 45 controls Canadian data Ewers, Am J Clin Nut 2008 Ebbert, Pediatric Transplantation 2015

Lisse, FASEB 2011 Vitamin D as a natural mtor inhibitor

Preliminary data from the CKD-MBD working group from the ESPN Chair: D. Haffner Board: S. Bakkaloglu, MA. Gamero, G. Reusz, R. Shroff Members: C. Pietrement, M.C. Matteucci, G. Di Zazzo, I. Guzzo, I. Durson, E. Petrosyan, O. Ozkaya, A. Anarat, F.L. Sever, G. Guido, S. Stabouli, G. Klaus, A. Prytula Liaison ESPN registry: M. Bonthius Liaison ESPN council: D. Haffner Liaison ERA-EDTA: J. Bacchetta Liaison ESCAPE network/4c-study: Franz Schaefer

Effect of vitamin D supplementation on bone/mineral metabolism and immune system in early CKD: a prospective observational cohort study* Working hypothesis Supplementation of vitamin D in children with early CKD (stage 2-3) has beneficial effects on surrogate markers of CKD-MBD Osteoblast activity (total and bone alkaline phosphatase) Osteoclast activity (TRAP5b, osteoprotegerin, RANK-L) Osteocyte activity (sclerostin) FGF-23, soluble klotho, 25OHD, calcitriol, 24,25-dihydroxyvitamin D, PTH *start-up funding: ESPN grant # 2014.01 *start-up funding: ESPN grant # 2014.01

Preliminary results of the pilot study * Aim Obtain information on sample size requirements and realistic endpoints for a future interventional trial Patients 4C-registry population (F. Schaefer) n=40 case-control study (vitamin D deficient +/- vit. D) randomized vitamin D trial GOSH (R. Shroff) n=40 ERGO Trial Preliminary data analysis: Christian Lerch *ESPN funding: grant # 2014.01

pmol/l Effects of vitamin D in CKD patients: 4C-study Preliminary data analysis: Christian Lerch

Vitamin D supplementation and bone biomarkers 4C GOSH 4C GOSH * * A significant increase of FGF23 and *p=0.03 SOST in patients receiving native vitamin D Preliminary data analysis: Christian Lerch

Vitamin D supplementation and soluble Klotho 4C GOSH * Reference values: 1460 ± 1300 pg/l (n=39) s-klotho deficiency defined as 765 pg/ml A significant increase of soluble Klotho *p=0.013 in patients receiving native vitamin D Preliminary data analysis: Christian Lerch

Conclusion and perspectives

Take-home points Vitamin D: native supplementation and active analogs A role for CKD-MBD of course, but also beneficial effects on Immunity Progression of renal disease Blood pressure Iron metabolism Guidelines tell us to correct vitamin D deficiency, but The next step will be to know how to correct it! The future challenge in pediatric nephrology Correcting 25-D levels: which target? Correcting 25- levels: which protocol? Daily, weekly, monthly, quarterly? Which dose? Which follow-up? How much will be too much?